As the processes for producing amino acids by using glutamic acid-producing coryneform bacteria belonging to the genus Corynebacterium or Brevibacterium, there have been known processes for producing glutamic acid by using wild-type strains of such genus and processes for producing various amino acids by using mutant strains derived from wild-type strains of such genus ["Amino Acid Fermentation, (I) and (II)" Association of Amino Acid and Nucleic Acid (Kyoritsu Shuppan, 1972); and "Amino Acid Fermentation" by Aida, Takinami and Chibata (Gakkai Publishing Center, 1986)]. Further, plasmid vectors for glutamic acid-producing coryneform bacteria [Japanese Published Unexamined Patent Application No. 134500/82 (European Patent No. 58889, U.S. Pat. No. 4617267), Japanese Published Unexamined Patent Application No. 183799/82 (European Publication No. 63763, U.S. Pat. No. 4500640) and Japanese Published Unexamined Patent Application No. 105999/83 (European Publication No. 82485, U.S. patent application Ser. No. 668674)], and methods for the transformation of these bacteria [Japanese Published Unexamined Patent Application No. 186492/82 (European Publication No. 63764, U.S. Pat. No. 4683205) and Japanese Published Unexamined Patent Application No. 186489/82 (European Publication No. 64680, U.S. Pat. No. 4681847)] have been recently developed. As a result, recombinant DNA technology has now become applicable to strains of the genus Corynebacterium or Brevibacterium, whereby new aspects on the effective utilization of these microorganisms have been opened.
The recombinant DNA technology has made it possible to clone a gene responsible for an enzyme that catalyzes the rate-limiting reaction in the biosynthetic pathway of an amino acid-producing microorganism and to introduce a recombinant DNA containing the cloned gene into a host microorganism, thereby enhancing the activity of said rate-limiting enzyme. There have been already known the cases in which amino acid productivity of coryneform bacteria has been increased by introduction of a recombinant DNA containing a cloned gene derived from a coryneform bacterium or from a microorganism of different species such as Escherichia coli [Japanese Published Unexamined Patent Application No. 126789/83 (European Publication No. 88166, U.S. patent application Ser. No. 787010), Japanese Published Unexamined Patent Application Nos. 156292/84, 156294/84, 24192/85, 34197/85, 30693/85 and 66989/85 (U.S. patent application Ser. Nos. 073888, 580814, 613209, 631648, 631649 and 646512) (European Publication No. 136359)]. Fermentation of these microorganisms of the genus Corynebacterium or Brevibacterium for the production of various amino acids is carried out by using, as the main carbon source, glucose, fructose, sucrose, maltose, acetic acid, ethanol, lactic acid or substances containing the same (for example, starch hydrolyzate and cane molasses), which these strains are hereditarily able to assimilate.
Attempts have also been made to use lactose as a carbon source for the production of amino acids by microorganisms belonging to the genus Corynebacterium or Brevibacterium. However, lactose cannot be employed as the main carbon source because the glutamic acid-producing coryneform bacteria have no ability to assimilate lactose [Nogeikagaku Kaishi, 39,328 (1965)]. The mixed culture is a known example of the process for producing amino acids by fermentation in which lactose is used as a carbon source. In the process, a lactose-assimilating bacterium (e.g., a lactobacillus) and an amino acid-producing bacterium belonging to the genus Corynbacterium or Brevibacterium are simultaneously cultured in a medium containing lactose, whereby lactose is assimilated into lactic acid by the former, and the latter assimilates lactic acid to produce glutamic acid, lysine or valine (Japanese Published Unexamined Patent Application Nos. 174095/82, 170194/82 and 208993/82).
Whey which is generated in large quantities as liquid waste from the cheese and casein manufacturing processes generally contains about 5% lactose and about 1% proteins. Although a part of it is used as food, animal feed additives and fertilizers, it is discarded for the most part. This is undesirable not only from the viewpoint of utilization of resources but also from that of environmental protection from pollution [The Food Industry, 18 No. 12, 20 (1975)]. Hence, use of whey as a fermentation material, if possible, would be of great industrial significance, as it would contribute to the effective utilization of resources and the environmental protection. The aforementioned mixed culture for the production of glutamic acid and other amino acids is intended for the utilization of lactose contained in whey, but it suffers various disadvantages. If lactic acid fermentation by the lactose-assimilating bacterium progresses rapidly, the culture broth becomes acidic, whereby the amino acid fermentation by the coryneform bacterium is inhibited. Accordingly, the amounts of the two strains to be inoculated and culture conditions must be elaborately determined according to the lactic acid-assimilating activity of the amino acid-producing strain belonging to the genus Corynebacterium or Brevibacterium which is used in the mixed culture. Thus, intricate operations and strict culture control are required. Under the circumstances, the present inventors tried to confer, by recombinant DNA technology, the ability to assimilate lactose on strains of Corynbacterium or Brevibacterium which genetically lack this ability in order to develop a new fermentation process for producing various amino acids in which a microorganism of Corynbacterium or Brevibacterium alone is cultured in a medium containing a lactose-containing substance such as whey as a carbon source.
This improvement of amino acid-producing bacteria by introduction of a recombinant DNA is based on the amplification effect of enzyme genes which are capable of expression in a coryneform bacterium. The expression of these genes takes place under the control of expression regulatory region inherent to the donor microorganism of these genes. Therefore, in some cases, the expression of the genes in a host microorganism may not be so efficient and the amino acid productivity can not be markedly improved. The expression efficiency of introduced genes must be enhanced in a suitable way to ensure a high amino acid productivity.
It is generally accepted that a promoter region necessary for the initiation of transcription and a ribosome-binding sequence essential to messenger RNA translation must be positioned upstream of a structural gene in order to ensure the expression of the gene in a microorganism, and that expression efficiency of the gene depends mainly on the activity of the promoter. Since the promoter region and the ribosome-binding sequence are considered to have species-specific base sequences, efficient expression of a desired structural gene would be achieved by disposing the structural gene downstream of a highly active promoter region and a proper ribosome-binding sequence derived from the host microorganism. Such an efficient gene expression system has already been established for Escherichia coli whose mechanism of gene expression has been reported in detail [Nakamura, K., Kagaku To Seibutsu, 20, 47 (1982)]and for Bacillus subtilis on which detailed genetic analysis has also been made [Horinouchi, S., Proteins/Nucleic Acids/Enzymes, 28, 1468 (1983); Goldfarb, D. S. et al., Nature, 293, 309 (1981)].
As a method for preparing a promoter region derived from a coryneform bacterium, there has been reported the use of a promoter-probing vector carrying a promoter-less chloramphenicol resistance gene which is used for detection of promoter activity (Japanese Published Unexamined Patent Application No. 124387/86). This vector contains the chloramphenicol resistance gene of Escherichia coli except its promoter region, that is, the structural gene and ribosome-binding sequence of that gene, and further has a promoter cloning site upstream therefrom. A DNA fragment containing the premoter region of a coryneform bacterium can be obtained by inserting a DNA fragment derived from the coryneform bacterium into this cloning site and selecting a recombinant plasmid which can confer chloramphenicol resistance on coryneform bacteria. It is considered that transcription of the chloramphenicol resistance gene in the recombinant plasmid thus obtained starts at the promoter region derived from the coryneform bacterium to form a messenger RNA, and translation of the messenger RNA is started by the function of the ribosome-binding sequence originated from the sequence of Escherichia coli chloramphenicol resistance gene, leading to the expression of the gene. However, the species-specificity in the gene-expression control region mentioned above suggests that insertion of a highly active promoter derived from a coryneform bacterium does not necessarily ensure efficient gene expression, because the ribosome-binding sequence of Escherichia coli might be unsuitable for translation in coryneform bacteria and hence translation might be rate-limiting in the expression process. Thus, it would be desirable to clone a DNA fragment having both a transcription initiation site and a translation initiation site in order to obtain a gene-expression control region which ensures efficient gene expression in coryneform bacteria. However, no effective method has so far been known to isolate such a DNA fragment.
It has been found that microorganisms belonging to the genus Corynebacterium or Brevibacterium can be imparted with the ability to assimilate lactose by introducing the genetic information derived from Escherichia coli lactose operon into a strain of the genus Corynbacterium or Brevibacterium by recombinant DNA technology and expressing the introduced information.
Further, intensive studies have been made to isolate the promoter region and the ribosome-binding sequence responsible for the initiation of transcription and translation, respectively, which are both essential to the expression of a gene in coryneform bacteria, from a chromosomal DNA. As a result, a plasmid vector has been constructed which has autonomous replicability in a coryneform bacterium, and contains a DNA fragment carrying structural genes coding for .beta.-D-galactosidase, or coding for .beta.-D-galactosidase and .beta.-galactoside permease derived from Escherichia coli lactose operon, and can be used to clone a DNA fragment responsible for the gene expression control in coryneform bacteria. The present invention has been accomplished based on these findings.